Multiple Enzyme Activities of Flavivirus Proteins

Abstract

© 2006 Novartis Foundation. All Rights Reserved. Dengue viruses (DENV) have 5′-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified ∼200 compounds having ≥50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5′-RNA triphosphatase activities. The NTPase and the 5′-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNAdependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito- or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3′ UTR and the RKRK motif in viral replication.